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CN111511289B - Method for determining ultraviolet sensitivity - Google Patents

Method for determining ultraviolet sensitivity Download PDF

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CN111511289B
CN111511289B CN201880082942.5A CN201880082942A CN111511289B CN 111511289 B CN111511289 B CN 111511289B CN 201880082942 A CN201880082942 A CN 201880082942A CN 111511289 B CN111511289 B CN 111511289B
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ultraviolet
irradiation
minutes
test substance
wave
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CN111511289A (en
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我部有
笠松慎也
小山内宰
武田胜也
中岛羊子
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Kao Corp
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Abstract

The present invention provides a method for noninvasively and rapidly determining sensitivity to ultraviolet rays. A method for determining ultraviolet sensitivity, comprising: and a step of irradiating the skin of the subject with ultraviolet light, and determining the ultraviolet sensitivity by using the amount of biophotons detected during a predetermined period after the irradiation, wherein 50% or more of the predetermined period overlaps with the period of 1 to 3 minutes after the irradiation.

Description

Method for determining ultraviolet sensitivity
Technical Field
The present invention relates to a method for determining ultraviolet sensitivity of human skin and a method for evaluating or selecting an ultraviolet sensitivity-reducing agent.
Background
Skin is subject to various damage from exposure to sunlight. In particular, it is known that: reactive Oxygen Species (ROS) generated in the skin by light in the ultraviolet region (290 to 400 nm) and biooxides generated by reaction with ROS exert deleterious effects on the skin as oxidative stress, and are closely related to erythema and pigmentation formation caused by short-term exposure, photoaging and carcinogenesis caused by long-term exposure, and the like (non-patent documents 1, 2).
As a technique for protecting skin from ultraviolet rays, there are the following methods: the skin is coated with sunscreen agent, antioxidant, etc., to inhibit ROS and organism oxide generated by ultraviolet irradiation, thereby preventing oxidative stress.
On the other hand, the living body has a system for eliminating active oxygen species and a countermeasure against an antioxidant substance in the living body against oxidative stress caused by external stimulus such as ultraviolet irradiation. However, these countermeasure means differ from one another in terms of age, genetic factors, lifestyle, eating habits, and the like, and the necessity of preventive care for ultraviolet rays varies from one person to another. Therefore, in order to effectively prevent and care ultraviolet rays, it is necessary to accurately evaluate oxidative stress generated in the skin by ultraviolet irradiation and the like and the antioxidant capacity in the skin.
As a method for evaluating oxidative stress of human skin, although there is a method using skin biopsy, it is invasive and therefore has not been widely used. Although the evaluation of oxidized proteins by peeling off the stratum corneum is low-invasive, it is difficult to say that the evaluation of oxidized proteins only remains in the stratum corneum and reflects the state of the inside of the skin (non-patent document 3). As a noninvasive evaluation method, there is a report of measuring carotenoids in the skin by raman spectroscopy, but this is an evaluation of single antioxidants, and it is difficult to say that the response of the whole skin is reflected (non-patent document 4).
Further, as a method for directly evaluating the sensitivity to oxidative stress due to ultraviolet irradiation, measurement of the minimum amount of ultraviolet rays (MED; minimum amount of erythema) that causes erythema after 24 hours of ultraviolet irradiation can be used as an indicator of the easiness of erythema caused by ultraviolet rays, but it is not easy to say that it is necessary to determine erythema formation after 24 hours of ultraviolet irradiation.
In this case, as a technique capable of noninvasively evaluating the oxidation state of a living body, a detection technique of weak luminescence of a living body (Biophoton) has been attracting attention. Biological photons are extremely weak spontaneous luminescence emitted by an organism as a result of vital activity. The reason for this is that singlet oxygen and excited carbonyl compounds are presumed, and luminescence due to oxidation reaction of a living body is considered.
Reported are: biophotons are observed in various living things such as plants, microorganisms, and animals, and in human skin, particularly, biophotons after irradiation with ultraviolet a waves (UVA) are measured, and the luminous intensity varies depending on the skin color (non-patent document 5), and the luminous intensity is reduced by applying an antioxidant cream (non-patent document 6).
However, these reports are evaluated by integrating values of several minutes immediately after UVA irradiation, and the details of oxidative stress response to time change are not evaluated with time, nor are they explicitly reported on what behavior biophotons show.
Prior art literature:
non-patent document 1: photodermotol Photoarmunol Photomed.18,75-81 (2002)
Non-patent document 2: toxicology 189,21-39 (2003)
Non-patent document 3: skin Res. Technology. 13,84-90 (2007)
Non-patent document 4: J.Invest. Dermatol.115,441-48 (2000)
Non-patent document 5: photodermotol. Photoarmunol. Photomed.25,65-70 (2009)
Non-patent document 6: skin Pharmacol. Physiol.24,300-4 (2011)
Disclosure of Invention
The present invention relates to the following 1) and 2).
1) A method for determining ultraviolet sensitivity, comprising: and a step of irradiating the skin of the subject with ultraviolet light, and determining the sensitivity of the subject by using the amount of biophotons detected during a predetermined period after the irradiation, wherein 50% or more of the predetermined period overlaps with the period of 1 to 3 minutes after the irradiation.
2) A method for evaluating or exploring an ultraviolet sensitivity-reducing agent, comprising: a step of administering or bringing a test substance into contact with a test object; and a step of irradiating the skin or skin cells of the subject with ultraviolet light, and evaluating the test substance by using the amount of biophotons detected in a predetermined period after the irradiation, wherein 50% or more of the predetermined period overlaps with the period of 1 to 3 minutes after the irradiation.
Drawings
Fig. 1 is a diagram showing the transition of the biophotonic amount after ultraviolet irradiation.
FIG. 2 is a graph showing the response biophotonic value after continuous application of aqueous rosemary solution.
Detailed Description
The present invention relates to an invention that provides a method for noninvasively and rapidly determining sensitivity to ultraviolet rays and a method for evaluating and exploring an ultraviolet sensitivity-reducing agent.
The inventors of the present invention studied a method that can evaluate skin damage caused by ultraviolet rays at an early stage, and as a result, found that a biophotonic agent detected within a predetermined period of time after irradiation of ultraviolet rays on human skin is closely related to formation of erythema occurring the next day, and that the sensitivity to ultraviolet rays can be evaluated and a material that reduces the sensitivity to ultraviolet rays can be evaluated and searched for using the amount of the biophotonic agent as an index.
According to the present invention, it is possible to contribute to the following measures: the method is used for noninvasively and rapidly judging the sensitivity to ultraviolet rays, and preventing erythema caused by ultraviolet rays from forming in the past, specifically, measures such as physical protection of ultraviolet rays, coating of ultraviolet ray-protecting skin external agents such as sun block (sun screen) and the like. Further, according to the present invention, the ultraviolet sensitivity reducing agent can be easily and efficiently evaluated or searched.
In the method for determining ultraviolet sensitivity of the present invention, ultraviolet light is irradiated to the skin of a subject, and the amount of biophotons detected within a predetermined period after the irradiation is used as an index.
In the method for evaluating or searching for an ultraviolet sensitivity-reducing agent of the present invention, when a test substance is administered to or brought into contact with the skin or skin cells of a subject, the skin or skin cells of the subject is irradiated with ultraviolet light, and the amount of biophotons detected within a predetermined period after the irradiation is used as an index.
In the method of the present invention, the ultraviolet ray to be irradiated is not particularly limited as long as it is Sup>A light ray having Sup>A wavelength in the ultraviolet region, and specifically, sup>A UV-B wave having Sup>A wavelength of 285 to 320nm or Sup>A UV-Sup>A wave having Sup>A wavelength of 320 to 400nm is given. In the present invention, the mixed ultraviolet rays of the a wave and the B wave are preferable, and the ratio of the intensity of light (a wave/B wave) is preferably 6 to 20, more preferably 7 to 15, still more preferably 8 to 12.
The intensity of the irradiated ultraviolet rays is not particularly limited, and is preferably 10mW/cm, for example 2 The above is more preferably 20mW/cm 2 The above is more preferably 30mW/cm 2 Above, and preferably 200mW/cm 2 Hereinafter, more preferably 170mW/cm 2 Hereinafter, it is more preferably 150mW/cm 2 The following is given. Further, it is preferably 10 to 200mW/cm 2 More preferably 20 to 170mW/cm 2 More preferably 30 to 150mW/cm 2 . In the present invention, the intensity of ultraviolet light means the intensity of ultraviolet light in Sup>A wavelength region (285 to 400 nm) where UV-B waves and UV-Sup>A waves are combined.
The intensity of ultraviolet light can be measured using a commercially available measuring instrument, and examples thereof include a Solarmeter Model 5.0 (uva+b) (Solartech inc.), a multifunctional spectroradiometer MSR-7000N (OptoResearch corporation), and the like.
The irradiation time varies depending on the intensity of the irradiated ultraviolet light, and may be, for example, 5 to 300 seconds, preferably 5 to 240 seconds, and more preferably 5 to 200 seconds.
The irradiation amount (irradiation energy) of ultraviolet rays is preferably 300 to 8000mJ/cm, which is determined by the irradiation intensity and irradiation time of ultraviolet rays 2 More preferably 500 to 7000mJ/cm 2 Further preferably 600 to 6000mJ/cm 2 。
The ultraviolet irradiation device for irradiating ultraviolet rays is not particularly limited as long as it has a light source capable of emitting light in the above wavelength range, and examples of the light source include a low pressure mercury lamp, a high pressure mercury lamp, an ultra-high pressure mercury lamp, a xenon lamp, a metal halide lamp, a wooden lamp, a fluorescent lamp, and the like, and a xenon lamp is preferable.
Such a light source can adjust the irradiation wavelength by combining filters capable of selecting light having a wavelength in the ultraviolet region, as necessary.
The skin area of the subject to be irradiated with ultraviolet light is not particularly limited as long as it can be irradiated with ultraviolet light and biophotonic measurement is possible, and the area is preferably a area which is less likely to be irradiated with living ultraviolet light because of the influence of daily ultraviolet light irradiation and fluctuation in ultraviolet sensitivity, which may cause variation in measurement values in the method of the present invention. Specifically, the skin such as the inner side of the forearm, the inner side of the upper arm, the back, the buttocks, and the abdomen is exemplified, and the inner side of the forearm, the inner side of the upper arm, the back, and the buttocks are preferable, and the inner side of the forearm or the abdomen is more preferable.
The subject to be examined in the method for evaluating or selecting ultraviolet sensitivity of the present invention may include, in addition to a human (subject), a cultured epidermal cell, a 3D skin model, a cultured skin tissue, or the like, and ultraviolet irradiation may be performed on a skin site of the subject as described above, or on the cell or tissue (skin) in the cultured epidermal cell or skin tissue. As the cultured epidermal cells, epidermal keratinocytes (keratinocytes) are preferable, and as the three-dimensional cultured skin cells, commercially available products such as EpiDermTM (manufactured by MatTek Corporation Co.), epiSkin (manufactured by Skinethic Co.), RHE (manufactured by Skinethic Co.), labcyte EPI model (manufactured by J-TEC Co.) and the like can be used.
As shown in test example 1 described below, the biophotons generated when ultraviolet light is irradiated to the skin show the maximum luminous intensity immediately after the ultraviolet light is irradiated, and then the luminescence decays with time. Further, according to the present invention, it was found that the luminous intensity mainly in the period of 1 to 3 minutes after the irradiation was correlated with the formation of erythema observed after the irradiation with ultraviolet rays for 24 hours (test examples 2 and 3).
Therefore, in the method for determining ultraviolet sensitivity or the method for evaluating or searching for an agent for reducing ultraviolet sensitivity according to the present invention, a period of 1 to 3 minutes after ultraviolet irradiation and a period in which 50% or more of the period overlaps with the period are set as predetermined periods, and the biophotonic amounts in the predetermined periods are used for determination or evaluation.
The predetermined period is set to a period in which 50% or more of the predetermined period overlaps with the period of 1 to 3 minutes after the irradiation, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more overlaps with the period of 1 to 3 minutes after the irradiation, and still more preferably 100% overlaps with the period of 1 to 3 minutes after the irradiation.
Here, the overlapping of the predetermined period and the period of 1 to 3 minutes after the irradiation means that there is a common period between the two periods, and the overlapping of 50% or more means that 50% or more of the predetermined period overlaps the period of 1 to 3 minutes after the irradiation.
Further, since the amount of photons in the initial period immediately after the irradiation of ultraviolet rays is large, the judgment or evaluation of the present invention is greatly affected, and therefore, the predetermined period of the present invention is preferably set so as not to overlap with the initial period immediately after the irradiation of ultraviolet rays. Specifically, the initial period from the end of irradiation to 15 seconds, preferably to 30 seconds, more preferably to 45 seconds is set so as not to overlap.
From the viewpoint of measuring an effective amount of biophotons, the length of the predetermined period is preferably 30 seconds to 3 minutes, more preferably 40 seconds to 2 minutes, still more preferably 50 seconds to 1 minute and 30 seconds, still more preferably 1 minute.
More preferable predetermined period includes, for example, 1 minute from 1 to 2 minutes after the irradiation, 1 minute from 2 to 3 minutes after the irradiation, and 2 minutes from 1 to 3 minutes after the irradiation.
The detection of the biophotonic is performed by an optical detection device including a detection unit such as a CCD having high sensitivity and low noise capable of detecting extremely weak biophotonic. As the optical detection device, for example, a weak light emission intensity detection device (CLA-IDFsk, manufactured by northeast electronics industry corporation) can be used. The wavelength of the detected emitted light varies depending on the photomultiplier tube of the detection device, and the device detects the photons of 300 to 850 nm. In order to minimize the influence of light from the measurement environment, it is preferable to perform the measurement of the biophotonic light in a space that is as light-shielded as possible, for example, in a darkroom.
That is, it is preferable that the ultraviolet irradiation device irradiates ultraviolet light to the measurement site in the darkroom, and then the biophotonic light emitted from the ultraviolet irradiation site is measured by the weak light emission intensity detection device. Further, the ultraviolet radiation section and the detection section in the weak light emission intensity detection device in the ultraviolet radiation device may be separated from each other, and from the viewpoint that ultraviolet radiation and bio-photon detection are possible without using a replacement device, it is preferable to integrate the ultraviolet radiation section (specifically, the optical fiber for light irradiation extending from the ultraviolet radiation device) and the detection section, and change the configuration of the device to be used by switching the optical paths.
The biophotonic light generated by ultraviolet irradiation is measured in advance for the luminous intensity at rest (also referred to as "steady-state biophotonic light"), then the luminous intensity within a predetermined period after ultraviolet irradiation (also referred to as "post-irradiation biophotonic light") is measured, and the value obtained by subtracting the luminous intensity at rest from this value is calculated as the luminous increment (also referred to as "response biophotonic light").
As described above, the degree of erythema observed 24 hours after uv irradiation was positively correlated with the amount of biophotons detected during the period of 1 to 3 minutes after uv irradiation. Therefore, the biophotonic amount can be used as an index for determining sensitivity to ultraviolet light or for evaluating or exploring an ultraviolet sensitivity-reducing agent. The "sensitivity to ultraviolet light" refers to the degree of an inflammatory reaction represented by the intensity or duration of an inflammatory reaction such as erythema caused by ultraviolet light, and the degree of an inflammatory reaction refers to the sensitivity to ultraviolet light. The term "decrease in ultraviolet sensitivity" means that the sensitivity to ultraviolet light is alleviated or suppressed, and the onset of inflammatory reactions such as erythema caused by ultraviolet light is suppressed.
In the ultraviolet sensitivity determination method of the present invention, for example, the amount of biophotons detected within a certain period after the ultraviolet irradiation of the present invention is measured for each age or age group, or for each sex, and obtained as basic data, and the age (age group) and sex deviation value of the subject can be calculated from the average value and standard deviation calculated therefrom as an index for determining the sensitivity to ultraviolet rays.
Further, regarding the determination index of the sensitivity to ultraviolet rays such as high sensitivity (easy redness), slightly high sensitivity (easy redness), standard, slightly low sensitivity (less redness), and the like, an appropriate evaluation criterion for associating these with the deviation value range is formulated, and based on this, the sensitivity to ultraviolet rays of the subject is determined from the deviation value of the subject.
The above-described method for determining ultraviolet sensitivity can perform determination in a very short time as compared with conventional MED measurement, and is less liable to be imposed on a subject. The information on the ultraviolet sensitivity obtained by the determination method of the present invention can be used as an index of product selection at the time of purchasing an ultraviolet protective skin external agent and product recommendation in the recommended sales of the ultraviolet protective skin external agent, for physical protection against ultraviolet rays or ultraviolet protection against ultraviolet rays by applying an ultraviolet protective skin external agent such as sun block (sun screen).
The method for determining ultraviolet sensitivity of the present invention corresponds to a method for collecting various data from a human body by measuring the structure, function, and the like of each organ of the human body, and is used for the above purpose. That is, the physical state or mental state such as the illness state or the health state of the person is not judged for medical purposes. In this sense, the method for determining ultraviolet sensitivity of the present invention may be also expressed as a method for measuring ultraviolet sensitivity or a method for checking ultraviolet sensitivity.
The evaluation or search of the ultraviolet sensitivity reducing agent of the present invention includes: and a step of administering or bringing the test substance into contact with the subject, wherein the skin or skin cells of the subject are irradiated with ultraviolet light, and the test substance is evaluated by using the amount of biophotons detected within a predetermined period after the irradiation.
The test substance to be administered is not particularly limited, and may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, or a compound, a composition, or a mixture thereof. Among them, a known substance ensuring safety, for example, a substance or a composition used as a raw material for pharmaceuticals, cosmetics, or the like is preferable. In addition, when the test substance is a composition such as a pharmaceutical or cosmetic, if the composition contains an ultraviolet protective material such as an ultraviolet absorber or an ultraviolet scattering agent, ultraviolet light irradiated to the skin or skin cells of the subject is physically protected in the ultraviolet irradiation of the evaluation or search method, and therefore the detected biophoton amount may be affected. Therefore, in the present evaluation or search method, when the test substance is a composition, it is preferable that the composition is a composition containing no ultraviolet ray protection material such as an ultraviolet ray absorber or an ultraviolet ray scattering agent. In the present evaluation or search method, when evaluating a composition containing an ultraviolet ray protection material as a test substance, it is preferable to perform a treatment of removing the composition from the skin or skin cells of a subject before the biophotonic measurement or before the ultraviolet ray irradiation.
The test substance may be administered orally or parenterally, preferably parenterally. Specifically, it is preferable to apply the composition to the skin in various forms such as ointments, creams, lotions, gels, aerosols, patches, tapes, and sprays.
In addition, the number of times the test substance is administered to the subject or the test substance is brought into contact with the subject is not particularly limited. Further, it is preferable that a predetermined period of time for the single administration or contact is provided before or simultaneously with the irradiation of the ultraviolet light, and the single administration or contact is performed at a predetermined frequency for the period of time. When the subject is a human, the administration period is preferably 1 day or more, more preferably 1 week or more, and still more preferably 4 weeks or more. Further, the time period is preferably 6 months or less, more preferably 3 months or less, and still more preferably 2 months or less. The administration frequency is preferably 1 or more times per day, more preferably 1 to 5 times per day, still more preferably 1 to 3 times per day, still more preferably 2 times per day.
When cultured epidermal cells, a 3D skin model, cultured skin tissues, or the like are used as the subject, the contact period is preferably 1 hour or more, more preferably 6 hours or more, and still more preferably 24 hours or more. Further, the time is preferably 72 hours or less, more preferably 36 hours or less, and still more preferably 48 hours or less. The contact frequency is preferably 1 or more times, more preferably 1 to 4 times, and still more preferably 1 or 2 times in the above-mentioned contact period.
Then, the biophotons detected during a predetermined period after ultraviolet irradiation were measured, and the test substance whose biophotons were reduced was evaluated as an ultraviolet sensitivity-reducing agent.
The identification of the test substance having a reduced biophotonic content can be performed, for example, by comparing the biophotonic content measured when different concentrations of the test substance are administered. As a more specific example, between a higher concentration test substance administration group and a lower concentration test substance administration group; between the test substance administration group and the placebo administration group; between the test substance administration group and the non-administration group; or comparing the biophotonic amounts before and after administration of the test substance. In the case of a reduction in the amount of biophotonic due to administration of a test substance, or due to administration of a higher concentration of a test substance, the test substance may be identified as a biophotonic amount-reducing substance.
For example, a test substance may be identified as a biophotonic amount-reducing substance when the biophotonic amount in the test substance administered group is considered to be prone to reduction, preferably considered to be statistically significantly reduced, as compared to the control group (placebo administered group or non-administered group).
Moreover, the identified test substance that reduces the amount of biophotons can be evaluated as an ultraviolet sensitivity reducing agent.
The ultraviolet sensitivity reducing agent selected in this way can be used, for example, as an external agent for skin for reducing ultraviolet sensitivity, or can be used in combination with an external agent for skin as a material or a preparation for reducing ultraviolet sensitivity.
With respect to the above embodiments, the present invention further discloses the following modes.
<1> a method for determining ultraviolet sensitivity, comprising: and a step of irradiating the skin of the subject with ultraviolet light, and determining the ultraviolet sensitivity by using the amount of biophotons detected during a predetermined period after the irradiation, wherein 50% or more of the predetermined period overlaps with the period of 1 to 3 minutes after the irradiation.
<2> the method according to <1>, wherein the predetermined period is preferably 30 seconds to 3 minutes, more preferably 40 seconds to 2 minutes, still more preferably 50 seconds to 1 minute, 30 seconds, still more preferably 1 minute.
<3> the method according to <1> or <2>, wherein the predetermined period is 1 minute from 1 to 2 minutes after the ultraviolet irradiation, 1 minute from 2 to 3 minutes after the ultraviolet irradiation, or 2 minutes from 1 to 3 minutes after the ultraviolet irradiation.
The method according to any one of <1> to <3>, wherein the ultraviolet irradiation is a mixed ultraviolet irradiation of an A wave and a B wave.
<5>Such as<1>～<4>The method according to any one of the above, wherein the ultraviolet irradiation is 300 to 8000mJ/cm 2 More preferably 500 to 7000mJ/cm 2 Further preferably 600 to 6000mJ/cm 2 Ultraviolet irradiation amount of (2).
The method of any one of <1> to <5>, wherein the amount of the biophotonic is calculated from the luminescence intensity of the biophotonic.
The method of any one of <1> to <6>, wherein the ultraviolet sensitivity is erythema formation caused by ultraviolet rays.
<8> a method for evaluating or searching for an ultraviolet sensitivity-reducing agent, comprising: a step of administering or bringing a test substance into contact with a test object; and a step of irradiating the skin or skin cells of the subject with ultraviolet light, and evaluating the test substance by using the amount of biophotons detected in a predetermined period after the irradiation, wherein 50% or more of the predetermined period overlaps with the period of 1 to 3 minutes after the irradiation.
<9> the method according to <8>, wherein the subject is a human, cultured epidermal cells, 3D skin model or cultured skin tissue.
The method of <8> or <9>, wherein a predetermined period of time for the administration or contact of the test substance to the subject is set before the irradiation of ultraviolet light, and one or more administrations or contacts are performed at a predetermined frequency of administration or contact during the period of time.
The method of <11> to <10>, wherein the administration period of the test substance to the subject is preferably 1 day or more, more preferably 1 week or more, more preferably 4 weeks or more, and preferably 6 months or less, more preferably 3 months or less, more preferably 2 months or less, and the administration frequency is preferably 1 day or more, more preferably 1 to 5 times, more preferably 1 to 3 times, more preferably 2 times.
<12> the method according to <10>, wherein, as the contact period, the contact of the test substance to the cultured epidermal cells, the 3D skin model or the cultured skin tissue is preferably 1 hour or more, more preferably 6 hours or more, more preferably 24 hours or more, and is preferably 72 hours or less, more preferably 36 hours or less, more preferably 48 hours or less, and the contact frequency is preferably 1 time or more, more preferably 1 to 4 times, more preferably 1 time or 2 times in the contact period.
Examples
Ultraviolet irradiation device
A WG-320 filter (thickness 1mm, manufactured by astringent valley optical company) was attached to a 300W type xenon light source (MAX-301, manufactured by daily spectroscopic company) having a xenon lamp as a light source, and was used (ultraviolet a wave (UVA): ultraviolet B wave (UVB) =10.8:1).
Test example 1Weak luminescence generated by ultraviolet irradiation to human
The subject was a healthy male aged 30 years old and was measured after 10 minutes of adaptation in a dark room. Measurement was performed using a weak light emission intensity detection device (CLA-IDFsk, manufactured by northeast electronics industry Co., ltd.) and the device was set in a quiet seatIs tightly attached to the inner side of the forearm. The detection unit fitting is integrally connected to an optical fiber for light irradiation from the ultraviolet irradiation device, and is configured to switch between ultraviolet irradiation and weak light emission intensity detection by switching the optical paths. Irradiating the modified part with ultraviolet (with ultraviolet intensity of 15.4, 32.5 or 61.6 mW/cm) 2 The irradiation time was 120 seconds, and the ultraviolet irradiation amounts were 1.8, 3.9 or 7.4J/cm, respectively 2 ) The light emission intensity was measured for 10 minutes from the end of irradiation for 2 seconds.
The measurement results are shown in fig. 1. It can be confirmed that: the amount of photons from the skin caused by ultraviolet irradiation reaches a maximum immediately after ultraviolet irradiation, and then decreases rapidly, and after a certain decrease, gradually decays with time.
Test example 2:weak luminescence measurement and erythema evaluation of human (1)
Ultraviolet irradiation and weak light emission intensity detection were performed in the same manner using the same apparatus as in test example 1. The subject was 3 healthy men aged 20 to 30 years old, and the detection unit fitting of the device was attached to the inner side of the forearm on a quiet seat. The emission intensity at rest was measured for 3 minutes before ultraviolet irradiation. Subsequently, ultraviolet irradiation was performed, and the light emission intensity was measured for 10 minutes from the end of irradiation for 2 seconds. The value obtained by subtracting the light emission at rest from the average value of the light emission at each time period is calculated as the light emission increment by the ultraviolet irradiation. The ultraviolet irradiation and the subsequent measurement of weak luminescence are performed under a plurality of ultraviolet irradiation conditions by changing the irradiation site. Specific irradiation conditions and measurement results are shown in table 1.
Further, regarding the formation of erythema at the ultraviolet-irradiated site, after 24 hours of irradiation, the skin color was measured using a high-speed spectrophotometer (CMS-35 FS, manufactured by color technology research, village) and evaluated for the difference in redness (Δa) between the irradiated site and the non-irradiated site in the vicinity of the irradiated site, and the results are shown in table 1.
From the results of table 1, a correlation coefficient (R) between Δa and the increase in luminescence due to ultraviolet irradiation was calculated (table 2).
TABLE 1
Figure BDA0002549836600000121
TABLE 2
Figure BDA0002549836600000131
*：p<0.05、**：p<0.01
Test example 3:weak luminescence measurement and erythema evaluation of human (2)
Ultraviolet irradiation and weak light emission intensity detection were performed in the same manner using the same apparatus as in test example 1. The subject was 10 healthy men aged 20 to 30 years old, and the detection unit fitting of the device was attached to the inner side of the forearm on a quiet seat. The light emission intensity at rest was measured for 3 minutes before ultraviolet irradiation. Subsequently, ultraviolet irradiation was performed, and the light emission intensity was measured for 5 minutes from the end of irradiation for 2 seconds. The value obtained by subtracting the light emission at rest from the average value of the light emission at each time period is calculated as the light emission increment by the ultraviolet irradiation. The ultraviolet irradiation and the subsequent weak luminescence measurement are performed under a plurality of ultraviolet irradiation conditions by changing the irradiation site. Specific irradiation conditions and measurement results are shown in table 3.
Further, regarding the formation of erythema at the uv-irradiated portion, the evaluation was performed as a difference in redness (Δa) in the same manner as in test example 2, and the results are shown in table 3.
From the results of table 3, a correlation coefficient (R) between Δa and the increase in luminescence due to ultraviolet irradiation was calculated (table 4).
TABLE 3
Figure BDA0002549836600000141
TABLE 4
Figure BDA0002549836600000142
**：p<0.01
As shown in tables 2 and 4, the amount of biophotons (response biophotons) detected during the 1 st to 2 nd and 2 nd to 3 rd minutes after uv irradiation had a significant positive correlation with the degree of erythema formation after uv irradiation.
Therefore, the sensitivity of the inflammatory reaction represented by the formation of erythema by ultraviolet light can be determined using the amount of biophotons detected mainly during the 1 st to 3 rd minutes after the ultraviolet light irradiation as an index.
Test example 4:evaluation of ultraviolet sensitivity reducing agent
1) The inspected person: 10 healthy adult Japanese men
2) Test article: 3.0% (v/v) Pharcolex Rosemary E aqueous solution
1.0% (v/v) Pharcolex Rosemary E aqueous solution
Placebo aqueous solution
* Concentration of Pharcolex Rosemary E (manufactured by Yi Bo Nature Mei Jian Co., ltd.) in aqueous solution as test substance
3) Response biophotonic assay and resolution:
the following steady-state biophotons and post-irradiation biophotons were measured, and the steady-state biophoton measurement value was subtracted from the post-irradiation biophoton measurement value during the period of 1 to 3 minutes after the end of light irradiation, to thereby calculate the response biophotons. For statistical analysis, a significant difference test was performed by single factor analysis of variance and multiple comparisons of Bonferroni.
< steady-state biophotons >
Weak luminescence of each examined region before ultraviolet irradiation was measured for 1 minute using a weak luminescence intensity detection device (CLA-IDFsk, manufactured by northeast electronics industry Co.).
< biological photon after irradiation >
The same ultraviolet irradiation apparatus as in test example 1 was used to irradiate 1430mJ/cm onto the examined region 2 The weak light emission of each test site immediately after the irradiation of ultraviolet light until 3 minutes was measured using a weak light emission intensity detection device.
4) The test steps are as follows:
the abdomen was rubbed with a wet paper towel, and 3 positions were marked as examined positions. The inspected portion was 5cm square (5X 5 cm). After 10 minutes of adaptation in the dark, biophotonic measurements were performed. Starting at night on the measurement day, 2mg/cm 2 The test pieces of (2) above were applied to the test sites at 3 positions for 4 weeks 1 day (early and late), respectively. After continuous use, biophotonic measurement was performed in the same step as before continuous use.
5) Results
The response bio-photon values after continuous use are shown in FIG. 2. Compared to placebo, the results were significantly reduced with rosemary (rosemary) 3.0% aqueous solution. In addition, it is known to inhibit the formation of erythema caused by ultraviolet irradiation by applying an extract of rosemary in advance (Japanese patent application laid-open No. 2002-87975).
Test example 5:evaluation of ultraviolet sensitivity-reducing agent (evaluation Using cultured cells)
1) And (3) cells: epidermal keratinocytes from normal human neonatal prepuce (frozen HEKn, lifeTechnologies)
(culture conditions)
According to the conventional method, in a proliferation medium (EpiLife, lifeTechnologies) for epidermal keratinocytes containing proliferation additives (Humedia-KG, kurabo), at 37℃and 5% CO 2 Is cultured under the condition of (2). In the weak luminescence assay, cells were recovered after washing with Hank's Balanced Salt Solutions (HBSS, invitrogen) balanced salt solution, and then washed with 4X 10 6 cells/ml were suspended in HBSS to provide a cell suspension.
2) Test article: trolox (6-hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid: 6-hydroxy-2,5,7, 8-tetramethylchromen-2-carboxilic acid) (Aldrich)
3) Determination of response biophotons:
the following steady-state biophotons and post-irradiation biophotons were measured, and the steady-state biophoton measurement value was subtracted from the post-irradiation biophoton measurement value during the period of 2 to 3 minutes after the end of irradiation, to thereby calculate the response biophotons. In addition, the biophotonic measurement and the ultraviolet irradiation were performed under the condition that the cover of the dish was removed.
< steady-state biophotons >
1.0ml of the cell suspension was placed in a 35mm dish, and the luminescence intensity before ultraviolet irradiation was measured in a dark room for 3 minutes using a weak luminescence intensity detection device (CLA-IDFsk, manufactured by northeast electronics industry Co.).
< biological photon after irradiation >
The same ultraviolet irradiation apparatus as in test example 1 was used to irradiate the solar ultraviolet simulator light (125 mW/cm 2 ) The light emission intensity after the irradiation was measured for 5 minutes at 40 seconds.
4) Test method
Trolox (final concentration 100 μm) or control solvent (70% ethanol) was added to normal human epidermal keratinocytes in 70-80% confluent state. Cells were recovered after 24 hours and the response biophotonic assays were performed according to the methods described above. The measurement data were evaluated relatively with the value of the control solvent group being 1.
5) Results
As shown in Table 5, in the Trolox-added group, which is a water-soluble analogue of vitamin E and known as an antioxidant, a tendency of inhibition in response to biophotonic intensity was confirmed (p < 0.1: no corresponding t test, both sides) compared to the control solvent group.
TABLE 5
Figure BDA0002549836600000171
(
Figure BDA0002549836600000172
p＜0.1)
Test example 6Evaluation of ultraviolet sensitivity-reducing agent (evaluation of skin Using tissue culture)
1) Skin tissue: normal human skin tissue from surgery was purchased from the united states skin depot National Disease Research Interchange (NDRI). In addition, in the tissue provision facility, written consent is given to the use of skin tissue for research purposes.
(culture conditions)
For human skin tissue, after trimming the subcutaneous fat, the skin was cut into small pieces of about 1cm×1cm in size using a 6-well plate at 37℃and 5% CO 2 Is cultured under the condition of (2). Advanced Dulbecco's Modified Eagle Medium (Life Technologies each) containing 10% (v/v) fetal bovine serum (Fetal Bovine Serum) (FBS) was used in the culture.
The skin after 1 day of culture was subjected to biophotonic assay.
2) Test article: trolox (6-hydroxy-2, 5,7,8-tetramethylchroman-2-carboxylic acid: 6-hydroxy-2,5,7, 8-tetramethylchromen-2-carboxilic acid)
3) Determination of response biophotons:
the following steady-state biophotons and post-irradiation biophotons were measured, and the steady-state biophoton measurement value was subtracted from the post-irradiation biophoton measurement value during the period of 1 to 3 minutes after the end of light irradiation, to thereby calculate the response biophotons. The biophotonic measurement and the ultraviolet irradiation were performed under conditions in which skin tissue was extracted from the culture medium.
< steady-state biophotons >
The light emission intensity before irradiation with the solar ultraviolet simulator was measured in a darkroom for 3 minutes using a weak light emission intensity detection device (CLA-IDFsk, manufactured by northeast electronics industry).
< biological photon after irradiation >
The same ultraviolet irradiation apparatus as in test example 1 was used to irradiate the solar ultraviolet simulator light (30 mW/cm 2 ) 3 minutes 20 seconds, controlThe emitted light intensity was measured for 5 minutes.
4) Test method
For cultured human skin tissue after 1 day of culture, trolox (final concentration 100 μm) or a control solvent (70% ethanol) was added to the culture medium, and after 24 hours, the skin tissue was washed with PBS, and then response biophotonic measurement was performed according to the above-described method. The measurement data were evaluated relatively with the value of the control solvent group being 1.
5) Results
As shown in Table 6, significant inhibition of the intensity of the response biophotons was confirmed in the Trolox-added group compared to the control solvent group (p < 0.05: no corresponding t-test, both sides).
TABLE 6
Figure BDA0002549836600000181
(*：p＜0.05)

Claims (27)

1. A method for judging ultraviolet sensitivity is characterized in that:
the method comprises the following steps: a step of irradiating the skin of the subject with ultraviolet rays, and determining the sensitivity of the ultraviolet rays by using the amount of biophotons detected during a predetermined period after the irradiation,
the predetermined period is a period in which 90% or more of the predetermined period overlaps with a period of 1 to 3 minutes after irradiation,
the ultraviolet sensitivity refers to the degree of inflammatory reaction caused by ultraviolet rays.
2. The method of claim 1, wherein:
the predetermined period is 30 seconds to 2 minutes.
3. A method according to claim 1 or 2, characterized in that:
the predetermined period is 1 minute from 1 to 2 minutes after the ultraviolet irradiation, 1 minute from 2 to 3 minutes after the ultraviolet irradiation, or 2 minutes from 1 to 3 minutes after the ultraviolet irradiation.
4. A method according to claim 1 or 2, characterized in that:
the ultraviolet irradiation is a mixed ultraviolet irradiation of a wave and B wave.
5. A method according to claim 1 or 2, characterized in that:
the ratio of the intensities of the light of the A wave and the B wave is 6 to 20 in terms of A wave/B wave.
6. A method according to claim 1 or 2, characterized in that:
the ultraviolet intensity of 285-400 nm in the wavelength region of A wave and B wave is 10-200 mW/cm 2 。
7. A method according to claim 1 or 2, characterized in that:
the ultraviolet irradiation time is 5 to 300 seconds.
8. A method according to claim 1 or 2, characterized in that:
the ultraviolet irradiation is 300-8000 mJ/cm 2 Ultraviolet irradiation amount of (2).
9. A method according to claim 1 or 2, characterized in that:
the amount of the biophotonic is calculated from the luminous intensity of the biophotonic.
10. A method according to claim 1 or 2, characterized in that:
the ultraviolet irradiation part is forearm inner part, upper arm inner part, back, buttock or abdomen.
11. A method according to claim 1 or 2, characterized in that:
uv sensitivity is the formation of erythema caused by uv light.
12. A method for evaluating or searching for an ultraviolet sensitivity-reducing agent, characterized by comprising:
the method comprises the following steps: a step of administering or bringing a test substance into contact with a test object; and a step of irradiating the skin or skin cells of the subject with ultraviolet light, and evaluating the test substance by using the amount of biophotons detected within a predetermined period after the irradiation,
the predetermined period is a period in which 90% or more of the predetermined period overlaps with a period of 1 to 3 minutes after irradiation,
the ultraviolet sensitivity refers to the degree of inflammatory reaction caused by ultraviolet rays.
13. The method as recited in claim 12, wherein:
the predetermined period is 30 seconds to 2 minutes.
14. The method of claim 12 or 13, wherein:
the predetermined period is 1 minute from 1 to 2 minutes after the ultraviolet irradiation, 1 minute from 2 to 3 minutes after the ultraviolet irradiation, or 2 minutes from 1 to 3 minutes after the ultraviolet irradiation.
15. The method of claim 12 or 13, wherein:
the ultraviolet irradiation is a mixed ultraviolet irradiation of a wave and B wave.
16. The method of claim 12 or 13, wherein:
the ratio of the intensities of the light of the A wave and the B wave is 6 to 20 in terms of A wave/B wave.
17. The method of claim 12 or 13, wherein:
the ultraviolet intensity of 285-400 nm in the wavelength region of A wave and B wave is 10-200 mW/cm 2 。
18. The method of claim 12 or 13, wherein:
the ultraviolet irradiation time is 5 to 300 seconds.
19. The method of claim 12 or 13, wherein:
the ultraviolet irradiation is 300-8000 mJ/cm 2 Ultraviolet irradiation amount of (2).
20. The method of claim 12 or 13, wherein:
the amount of the biophotonic is calculated from the luminous intensity of the biophotonic.
21. The method of claim 12 or 13, wherein:
the subject is a human, cultured epidermal cells, a 3D skin model, or cultured skin tissue.
22. The method of claim 12 or 13, wherein:
in the case of the administration or contact of a test substance to a subject, a predetermined administration or contact period is set before the irradiation of ultraviolet rays, and one or more administrations or contacts are performed at a predetermined administration or contact frequency during the period.
23. The method as recited in claim 22, wherein:
the administration period of the test substance to the subject is 1 day or more and 2 months or less.
24. The method as recited in claim 22, wherein:
the test substance is administered to the subject 1 to 5 times per day.
25. The method as recited in claim 22, wherein:
the contact period of the test substance with the cultured epidermal cells, the 3D skin model or the cultured skin tissue is 1 to 48 hours.
26. The method as recited in claim 22, wherein:
the contact frequency of the test substance to the cultured epidermal cells, the 3D skin model or the cultured skin tissue is 1 to 4 times.
27. The method of claim 12 or 13, wherein:
between a higher concentration of test substance administration or contact group and a lower concentration of test substance administration or contact group; between the test substance administration or contact group and the placebo administration or contact group; between the test substance administered or contacted and non-administered or non-contacted groups; or comparing the biophotonic amounts before and after administration or contact with the test substance, and identifying the test substance as a biophotonic amount-reducing substance when the biophotonic amount is reduced due to administration or contact of the test substance at a higher concentration or administration or contact of the test substance.
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